Amplite™ Fluorimetric Sphingomyelinase Assay Kit *Red Fluorescence*

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1e+4100010010.1- Dose-responseData legend Generated with Quest Graph™ Sphingomyelinase (mU/mL) RFU Hover mouse to interact
Sphingomyelinase dose response was measured on a solid black 96-well plate with Amplite™ Fluorimetric Sphingomyelinase Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).
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200 Tests 13621 $195


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
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Overview

Ex/Em (nm)571/585
Storage Freeze (<-15 °C)
Minimize light exposure
InstrumentsFluorescence microplate reader
Category Enzyme Detection
Phosphodiesterases
Related
Five types of sphingomyelinase (SMase) have been identified based on their cation dependence and pH optima of action. They are lysosomal acid SMase, secreted zinc-dependent acid SMase, magnesium-dependent neutral SMase, magnesium-independent neutral SMase, and alkaline SMase. Among the five types, the lysosomal acidic SMase and the magnesium-dependent neutral SMase are considered major candidates for the production of ceramide in the cellular response to stress. Our Amplite™ Fluorimetric Sphingomyelinase Assay Kit provides the most sensitive method for detecting neutral SMase activity or screening its inhibitors. The kit uses Amplite™ Red as a fluorogenic probe to indirectly quantify the phosphocholine produced from the hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). It can be used for measuring the SMase activity in blood, cell extracts or other solutions. The fluorescence intensity of Amplite™ Red is proportional to the formation of phosphocholine, therefore to the SMase activity. Amplite™ Red enables the assay readable either in fluorescence intensity or absorption mode. The kit is an optimized "mix and read" assay that can be used for real time monitoring of Smase activities. Our kit 13622 has been developed for monitoring acid SMase activity.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare Sphingomyelin working solution (50 µL)
  2. Add SMase standards and/or SMase test samples (50 µL)
  3. Incubate at 37°C for 1 - 2 hours
  4. Add Sphingomyelinase working solution (50 µL)
  5. Incubate at RT for 1 - 2 hours
  6. Monitor fluorescence intensity at Ex/Em = 540/590 nm (Cutoff = 570 nm)

Important notes
Thaw one vial (or bottle) of each kit component at room temperature before starting your experiment.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:540 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
Preparation of stock solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Sphingomyelinase standard solution (10 U/mL):
Add 20 µL of PBS with 0.1% BSA into the vial of Sphingomyelinase Standard (Component F) to make a 10 units/mL Sphingomyelinase standard solution.

2. Amplite™ Red stock solution (200X):
Add 80 µL of DMSO (Component G) into the vial of Amplite™ Red (Component C) to make 200X Amplite™ Red stock solution. Keep from light. Note: The Amplite™ Red is unstable in the presence of thiols (such as DTT and 2-mercaptoethanol). The final concentration of DTT or 2-mercaptoethanol in the reaction should be lower than 10 µM. Amplite™ Red is also unstable at high pH (>8.5). The reactions should be performed at pH 7 - 8. pH 7.4 is recommended for the assay buffer.

 

Preparation of standard solution
Sphingomyelinase standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/13621

Add 1 µL of 10 units/mL Sphingomyelinase standard solution into 1000 µL Assay Buffer (Component E) to generate a 10 mU/mL Sphingomyelinase standard solution. Take 10 mU/mL Sphingomyelinase standard solution and perform 1:2 serial dilutions to get serially diluted Sphingomyelinase standards (SMase7 - SMase1). Note: Diluted Sphingomyelinase standard solution is unstable. Use within 4 hours.

Preparation of working solution

1. Sphingomyelin working solution:
Add 50 µL of Sphingomyelin (Component B) into 5 mL of SMase Reaction Buffer (Component D) and mix well to make Sphingomyelin working solution. Note: The Sphingomyelin working solution should be used promptly.

2. Sphingomyelinase working solution:
Add 5 mL of Assay Buffer (Component E) into the bottle of Enzyme Mix (Component A) and mix them well. Then, add 25 µL of 200X Amplite™ Red stock solution into the bottle of Enzyme Mix solution to make Sphingomyelinase working solution before starting the assay. Note: The Sphingomyelinase working solution should be used promptly and kept from light; longer storage is likely to cause high assay background.

Sample experimental protocol

Table 1. Layout of Sphingomyelinase standards and test samples in a solid black 96-well microplate. SMase = Sphingomyelinase Standards (SMase1 - SMase7, 0.078 to 5 mU/mL), BL = Blank Control, TS = Test Samples. 

BL BL TS TS
SMase1 SMase1 ... ...
SMase2 SMase2 ... ...
SMase3 SMase3    
SMase4 SMase4    
SMase5 SMase5    
SMase6 SMase6    
SMase7 SMase7    

Table 2.  Reagent composition for each well.

Well Volume Reagent
SMase1 - SMase7 50 µL Serial Dilutions (0.078 to 5 mU/mL)
BL 50 µL Assay Buffer
TS 50 µL test sample
  1. Prepare Sphingomyelinase standards (SMase), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL. Note: Treat your cells or tissue samples as desired.

  2. Add 50 µL of Sphingomyelin working solution to each well of Sphingomyelinase standard, blank control, and test samples to make the total Sphingomyelin assay volume of 100 µL/well. For a 384-well plate, add 25 µL of Sphingomyelin working solution into each well instead, for a total volume of 50 µL/well.

  3. Incubate the reaction mixture at 37°C for 1 - 2 hours.

  4. Add 50 µL of Sphingomyelinase working solution to each well of Sphingomyelinase standard, blank control, and test samples to make the total Sphingomyelinase assay volume of 150 µL/well. For a 384-well plate, add 25 µL of Sphingomyelinase working solution into each well instead, for a total volume of 75 µL/well.

  5. Incubate the reaction mixture for 1 - 2 hours at room temperature (protected from light).

  6. Monitor the fluorescence increase with a fluorescence microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm).
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Sphingomyelinase samples. We recommend using the Online Linear Regression Calculator which can be found at:

https://www.aatbio.com/tools/linear-logarithmic-semi-log-regression-online-calculator

Figure 1. Sphingomyelinase dose response was measured on a solid black 96-well plate with Amplite™ Fluorimetric Sphingomyelinase Assay Kit using a Gemini fluorescence microplate reader (Molecular Devices).

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References & Citations

Doxepin mitigates noise induced neuronal damage in primary auditory cortex of mice via suppression of acid sphingomyelinase/ceramide pathway
Authors: Yu-Ting Su, Xing-Xing Meng, Xi Zhang, Yi-Bin Guo, Hai-Jun Zhang, Yao-Ping Cheng, Xiao-Ping Xie, Yao-Ming Chang, Jun-Xiang Bao
Journal: The Anatomical Record (2017)

New Aspects of Silibinin Stereoisomers and their 3-O-galloyl Derivatives on Cytotoxicity and Ceramide Metabolism in Hep G2 hepatocarcinoma Cell Line
Authors: Mahdi Mashhadi Akbar Boojar, Shahram Ejtemaei Mehr, Mahsa Hassanipour, Masoud Mashhadi Akbar Boojar, Ahmad Reza Dehpour
Journal: Iranian Journal of Pharmaceutical Research (2016): 421--433

Riccardin DN induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo
Authors: Lin Li, Huanmin Niu, Bin Sun, Yanan Xiao, Wei Li, Huiqing Yuan, Hongxiang Lou
Journal: Toxicology and Applied Pharmacology (2016): 175--184

Mitochondrial respiration controls lysosomal function during inflammatory T cell responses
Authors: Francesc Baixauli, Rebeca Acín-Pérez, Carolina Villarroya-Beltrí, Carla Mazzeo, Norman Nunez-Andrade, Enrique Gabandé-Rodriguez, Maria Dolores Ledesma, Alberto Blázquez, Miguel Angel Martin, Juan Manuel Falcón-Pérez
Journal: Cell metabolism (2015): 485--498

The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism
Authors: Warren Davis
Journal: Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids (2014): 168--179

A high-throughput sphingomyelinase assay using natural substrate
Authors: Miao Xu, Ke Liu, Noel Southall, Juan J Marugan, Alan T Remaley, Wei Zheng
Journal: Analytical and bioanalytical chemistry (2012): 407--414






Additional Documents

 
Safety Data Sheet (SDS)


Catalogs
1. Enzyme Probes & Assay Kits

Application Notes
1. AssayWise Letters 2014, Vol 3(1)

Certificate of Analysis